Vitamin A in Epithelial Differentiation and Skin Carcinogenesis

Februafy 1994: (IIS45-552

Vitamin A in Epithelial Differentiation and Skin Carcinogenesis Luigi M. De Luca, Ph.D., Nadine Darwiche, Ph.D., Giulia Celli, B.S., Karolina Kosa, Ph.D., Carol Jones, M.S., Sharon Ross, Ph.D., and Li-Chuan Chen, Ph.D.

Epithelial Phenotypes

The very existence of an organism, from conception through development to normal functioning of the adult, depends on vitamin A. To think that all these stages require vitamin A because of its action on epithelial tissues is probably simplistic. Neverthe- less, it seems clear that the most easily observable alterations in the vitamin A-deficient animal take place in epithelial tissues.

These are, in general, tissues that cover internal and external body surfaces that come in direct con- tact with the outside environment and, therefore, with air and its pollutants or food and its contami- nants. Epithelia are differentially structured depend- ing on their locations and functions. Major epithe- lial phenotypes recognizable in order of their increasing complexity in histology (Figure 1) are ( 1 ) the simple columnar epithelium, characterized by one layer of columnar cells, all in contact with the basement membrane, for example, the endo- cervical epithelium and intestinal mucosa; (2) the pseudostratified epithelium, in which basal and co- lumnar cells share the basement membrane, as found in tracheobronchial epithelium; (3) the squa- mous stratified epithelium, in which several cell lay- ers, basal and suprabasal (squamous), form a barrier to mechanical and air impact, for example, the vagi- nal and ectocervical epithelia, this phenotype can be converted to (4) the squamous stratified keratinizing epithelium usually found in the epidermis.’

These four epithelial phenotypes are found in spe- cific locations because they serve specific functions.

Drs. De Luca, Darwiche, Celli, Kosa, Jones, Ross, and Chen are Section Chief (LDL), Postdoctoral Fel- low (ND, KK, SR, and LC), and Research Biologist (GC and CJ) at the Differentiation Control Section, Laboratory of Cellular Carcinogenesis and Tumor Pro- motion, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Their presence, however, is subject to the regulation of hormonal cycles and the nutritional intake of vi- tamin A. The most intriguing example of the way epithelial phenotypes are governed by these factors is the cervical epithelium, in which two distinct pheno- types are expressed at specific locations. For example, the squamo-columnar junction of the cervical epithe- lium is joined by the squamous stratified epithelium of the cervix uteri located below the junction, and, thus, termed subjunctional, and the simple columnar epithelium located above the junction, and therefore termed suprajunctional (Figure 2). The squamo-co- lumnar junction moves toward the endocervix or ec- tocervix throughout life, depending on hormonal and functional status. The squamo-columnar junction is of interest to cancer researchers because it is the site for carcinoma development.2.” Later in this article we will return to the scheme of epithelial phenotypes (Figure 1) to reflect on their interchangeability as induced by retinoid status and the action of certain hormones (e.g., steroid hormones).

Vitamin A Deficiency and the Formation of Squamous Metaplastic Lesions

Epithelial cancer is the most common form of neo- plastic disease. Skin, lung, colon, breast, prostate, cervical, bladder, and esophageal cancers arise in epithelial tissues and acquire the ability to grow and invade through the basement membrane. A common preneoplastic lesion precursor to all squamous cell carcinomas arising in these epithelia is squamous metaplasia.””

To characterize the formation of squamous metaplasia in the columnar epithelium of the cervix uteri, we studied the keratin expression profile. Dar- wiche et al.I2 found that the subjunctional squa- mous-stratified epithelium usually expresses several keratins (e.g., K5/K14, Kl/K10, K6, and K13) not normally found in the columnar epithelium supra- junctionally (data not shown and Figure 3A). Con-

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Figure 1. Influence of retinoid status on epithelial phenotypes. The chart schematically represents the phenotypic con- version caused by vitamin A deficiency in the endocervical and tracheal epithelia. A simple columnar epithelium (e.g., the endocervical epithelium) is gradually converted to a stratified keratinizing phenotype resembling the epidermis under severe vitamin A-deficient conditions. Similarly, a pseudostratified epithelium (e.g., the tracheal epithelium) is gradually transformed into a stratified epidermoid keratinizing epithelium. These changes are fully reversible in the presence of retinoic acid (RA). Reproduced from Rosenthal et al.,’ with permission.

versely, the simple columnar epithelium specifically expresses keratins (e.g., K8) not found in the squa- mous stratified epithelium (Figure 3B). A mild de- ficiency of dietary retinoic acid (RA) permits K5 immunohistochemical staining of single basal-like cells, called “reserve cells,” in the suprajunctional location (Figure 3C). These reserve cells grow and eventually form squamous metaplastic foci, which are found in locations proximal and distal to the junction (Figure 3D). This suggests that the reserve cells and the squamous foci may not “migrate” in a sub- to suprajunctional direction, but rather are formed in a subcolumnar position on the basal membrane. The condition of severe vitamin A de- ficiency permits complete replacement of the simple columnar epithelium by a stratified squamous epi- thelium in continuity with the ectocervical pheno-

type (Figure 3E). This also permits keratinization of some of the uterine glands with disappearance of the keratin K8 (Figure 3F). These changes are de- tectable at the mRNA level by in situ hybridiza- tion.’* Under normal conditions, K5 mRNA is strict- ly expressed in the subjunctional squamous stratified epithelium, as shown in Figure 4A-D. Su- prajunctional squamous metaplastic epithelium also shows K5 mRNA expression (Figure 4E,F).

From these studies and other pertinent work we suggest the following concepts:

1. RA is essential for the maintenance of specific phenotypes, as demonstrated by the keratin pro- file in the epithelial tissues.

2. The possible interaction of steroid hormones and retinoids and their respective nuclear receptors

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Figure 2. Schematic representation of the squamo-columnar junction (SCJ) with stratified squamous (subjunctional) epithelium and simple columnar (suprajunctional) epithelium. Adapted with permission from Ca~tier.'~

3.

4.

controls the expression of epithelial phenotypes and permits the existence and adjacency of squa- mous stratified and simple columnar epithelia at the squamo-columnar junction. Basal-like reserve cells and squamous metaplas- tic foci form de novo in a subcolumnar position. These cells synthesize a family of keratins usu- ally present in the squamous stratified epithelium of the ectocervix. Exposure to chemical carcinogen^,'^ viral a g e n t ~ , ' ~ or both favors formation of squamous metaplastic lesions. Dietary retinoids are neces- sary for replacement of the squamous lesions by the normal columnar epithelium.

Retinoic Acid Receptor Expression in Different Epithelial Phenotypes

Retinoid research at the molecular level has ad- vanced greatly in recent years, mainly because of the discovery of nuclear receptors for all-trans and 9 4 s retinoic Six genes code for the pro- teins RARa, p. and y and RXRa, p. and y. The use of two different promoters (P1 and P2) in these genes and/or alternative mRNA splicing are respon-

sible for 18 isoforms of the RARs and probably several, as yet undetermined, RXR isoforms.'8

Specific probes, kindly provided by Drs. Pierre Chambon, Strasbourg, France, and by Ronald Evans, La Jolla, CA, USA, were used in our laboratory to study mRNA expression of these receptors in epi- thelial tissues displaying the squamous stratified and the columnar epithelial phenotypes (Darwiche et al., submitted). RARa major isoforms are ubiquitously expressed basally and suprabasally in squamous stratified (Figure 5A), squamous metaplastic, and co- lumnar epithelia (Figure 5B). In sharp contrast RARp major isoforms are only expressed in colum- nar epithelium and basal cells of squamous meta- plastic foci (Figure 5C,D). It is of interest to note that RARp receptors were also found in the basal cells of squamous metaplastic foci in continuity with the columnar cells (Figure 5D), indicating that these basal cells may be related to columnar cells. In con- trast, basal cells of the ectocervical squamous strat- ified epithelium do not express the RARp receptor, but do express RARy. Therefore, these two popula- tions of basal cells are not identical, and we speculate that the basal cells of the squamous metaplastic le- sion possess the potential to give rise to columnar

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Figure 3. Immunohistochemistry of keratins K5 (A,C,E) and K8 (B,D,F) in cervical sections of mice. (A,B) Retinoic acid (RA) + diet; (C,D) mild vitamin A deficiency; (E,F) severe vitamin A deficiency. All enlargements are X50-fold. Reproduced from Darwiche et al.,” with permission.

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Figure 4. In situ hybridization of K5 mRNA in cervical sections of mice. (A-D) Retinoic acid (RA) + diet; (E,F) severe vitamin A deficiency. (A,B) At squamo-columnar junction, (C) subjunctional epithelium, (D-F) suprajunctional epithe- lium (X200). Reproduced from Darwiche et al.,'' with permission.

cells when RA is made available. However, supra- basal cells of the squamous lesion do not express RARP, and in this aspect they are similar to the cells of squamous stratified epithelium.

It is our aim to study the expression of RXRa, P, y, and the estrogen receptor to understand pos- sible distinct or interactive roles of these receptors and the RARs. It is also our intention to study re- ceptor expression in squamous cell carcinoma to in- vestigate further the histogenetic mechanisms re- sponsible for the formation of this tumor.

Retinoic Acid as a Chemopreventive Agent of Skin Carcinoma Formation

Skin carcinogenesis can be divided into at least three stages: initiation, promotion, and malignant conver~ion.'~ De Luca et al.*O recently reported a strong inhibition in the conversion of papillomas to carcinomas in the back skin of female SENCAR mice by dietary retinoic acid at the pharmacological dose of 30 pg/g of diet. Dietary RA at this dose did not inhibit the promotion stage to any significant

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Figure 5. In situ hybridization of RARa2 mRNA (A,B) and RARP3 mRNA (C,D) in cervical sections of mice. (A,C) Subjunctional epithelium; (B,D) severe vitamin A-deficient suprajunctional epithelium. All enlargements are (X200), except for D (X400).

extent. This is in contrast to the results of topical application of RA to SENCAR mice treated with 7,12-dimethylbenz[a]anthracene (DMBA) and 12- 0-tetradecanoylphorbol- 13-acetate (TPA) in a sim- ilar protoco1.2'.22 The protocol of a two-stage chemical carcinogenesis using DMBA as the initi- ator and TPA as the tumor promoter is illustrated in Figure 6. This system permits the development of multiple benign lesions (papillomas), starting at about 11-12 weeks of age, which is about 8-9 weeks after initiation. Most mice develop these be-

nign lesions at about week 18 of age. When TPA application is stopped at week 23, papillomas re- gress. Carcinomas, however, begin to appear at about week 20. Conversion efficiency from papil- lomas to carcinomas is shown in Table 1. Con- sumption of a diet with 30 pg retinoic acid per gram blocks carcinoma formation (Table 1). Interestingly, dietary p-carotene (600 pg/g of diet) also inhibits carcinoma formation; however, the effect of p-car- otene on papilloma formation is variable, depending on the dose, and other unidentified fact0rs.2~

INITIATION PROMOTION CONVERSION DMBA 2Opg at wk 3 TPA 2pghnrk weekly from

wk4 to 23

1 I I Normal Cell - Initiated Cell - Papilloma ---+ Carcinoma Figure 6. Scheme of a typical two-stage skin chemical carcinogenesis protocol.

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Since alterations in RAR genes have been dem- onstrated in certain cancerous cells, particularly from patients with acute promyelocytic leukemia (APL),24-27 we used in situ hybridization to analyze the expression of six major isoforms, RARa1,2, RARP2,3, and RARy1,2 (Darwiche et al., manu- script in preparation). We found that RARy1 and 2 isoforms are both expressed in normal, papilloma, and carcinoma tissue, whereas RARal and 2 iso- forms are either decreased or absent from carcinoma tissues, although both isoforms are highly expressed in normal skin and in papillomas. In contrast, RARPs are not expressed in the skin.

Summary and Conclusions

Our concepts on epithelial phenotypes are illustrated in Figure 1, which summarizes the multiplicity of the principal epithelial phenotypes under normal nutri- tional intake of vitamin A. These phenotypes can be (1) simple columnar, as found in the endocervix; (2) pseudostratified, as for the trachea; (3) squamous stratified, as for the ectocervix; and (4) keratinizing squamous stratified, as for the epidermis. Nutritional deficiency of vitamin A permits transformation of the simple columnar endocervical epithelium, initially into a pseudostratified epithelium as found in trachea by the appearance of basal-like “reserve” cells, then into the squamous stratified phenotype, and eventu- ally into the keratinizing squamous stratified pheno- type. Obviously, nutritional as well as hormonal fac- tors are involved in the definition of morphological characteristics of the various lining epithelia of the body. We are studying the interplay between RA and steroid hormones in the development of these changes.

In situ hybridization revealed a specific epithelial localization of the RARs. In particular, RARP iso- forms were found to be localized in the columnar epithelium, suprajunctionally, and RARy isoforms in the squamous stratified epithelium, subjunctionally. RARa isoforms were found distributed throughout the stratified squamous and the simple columnar phe- notypes. The interplay or differential expression of specific RARs, and possibly of RXRs and estrogen receptors in the cervical epithelium, may be an im- portant determinant of the differentiated phenotype. Our present efforts are aimed at understanding the regulation of receptor expression in normal, preneo- plastic, and neoplastic cervical and skin epithelia. Preliminary results indicate a significant down-reg- ulation of RARa, specifically in skin carcinomas. Whether this is a consequence or a cause of malig- nant conversion remains to be determined.

A second item of interest is that dietary RA, in pharmacologic doses, inhibits conversion of the be- nign lesion papilloma to carcinoma without influenc-

Table 1. Conversion Efficiency of Papillomas to Carcinomas

Conver- sion

Dietary Papilloma Carcinoma Efficiency* Groud No. No. (%)

RA 0.3 pg 260 53 1.9 RA 3.0 pg 259 5’ 1.9 RA 30.0 pg 320 0 0

I The initial numbers of mice for the three dietary groups, ranging from 0.3 to 30 pg of RA, were 20, 19, and 25, respectively. The numbers of mice with the maximum papillomas were 20, 16, and 25, respectively. The num- bers of mice alive when the first carcinoma appeared were 14, 13, and 22, respectively. ’ The conversion efficiency was the total number of car- cinomas divided by the maximum number of papillo- mas X 100.

Five mice from each group developed carcinomas.

ing papilloma formation to any significant extent. A likely hypothesis to explain this effect is that RA up- regulates retinoid receptor expression, and that this receptor protein might form complexes with AP-1 proteins, such as c-fos, thereby preventing their ac- tion in malignant conversion. This hypothesis is be- ing investigated in our laboratory and is particularly relevant, since c-fos has been shown to cause malig- nant conversion of papilloma to carcinoma cells in grafting Alternatively, RA may induce differentiation and cell loss in papilloma and/or car- cinoma cells, thereby reducing the chance for the formation of visible malignant lesions. This latter mechanism has already been demonstrated in the in- duction of differentiation of APL cells in vivo and in vitr~.~’~’

The retinoids represent a family of compounds that is uniquely capable of interacting with processes as diverse and yet interconnected as epithelial phe- notypic interconversions and benign to malignant neoplastic transformation. This versatility is reflected in their use as differentiation agents in APL.32 The work of our laboratory is presently focused on un- derstanding the steps involved in the neoplastic pro- cess and developing clinical strategies to control neo- plastic progression.

Acknowledgements. This manuscript was pre- pared in part while Luigi M. De Luca was a visiting professor in the First Department of Internal Medi- cine, University of Gifu, Japan. This author thanks Yukio and Etsuyo Nakamachi for their kind hospi- tality.

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